Beta Vulgaris root plasma membrane vesicles show a very high water permeability, sensitive to pH but not to phosphorylation

. KARINA ALLEVA, MOIRA SUTKA, RICARDO DORR, MARIO PARISI Y GABRIELA AMODEO.

Cuernavaca, México

Congreso; 1st Pan-American Plant Membrane Biology Workshop; 2003

Comité organizador ad hoc

Despite the high abundance of aquaporins in the plasma membrane of plant cells, the first measurements of water permeability (Pf) in vesicles showed low values of Pf associated to a high activation energy (1, 2). Gerbeau et al (3) developed a procedure for the purification of plasma membrane vesicles (PMV) using a cocktail of phosphatases inhibitors and divalent cations chelators and under these conditions they obtained PMV with higher Pf indicating the presence of active water channels. In the present work right side-out PMV from red beet roots (Beta vulgaris) were purified in two different conditions, one with cation chelators and phosphatase inhibitors and other lacking this kind of protection. To measure water transport, PMV were first pre-equilibrated and then an inwardly directed osmotic gradient was applied by increasing the osmolarity of the external solution. Volume changes were followed by light scattering (90º, 500 nm) in a stopped-flow spectrophotometer and Pf was calculated according to (XX). The Pf value obtained in standard conditions  (261±42 mm s-1 (±SEM, n=7)), was not different from the one resulted under protected conditions (217±30 mm s-1 (±SEM, n=3)). This permeability coefficient, is very high in comparison with published values for other plant species (4, 5) including PMV obtained by protected cocktails. This observation among the presence of a low activation energy and inhibition by HgCl2 points to the presence of active water channels in Beta vulgaris roots plasma membrane. Interestingly, acidic pH in the interior of vesicles and some divalent cations, mainly calcium, are capable of strongly reduce the water permeability. This work presents therefore novel contributions to the characterization of Beta vulgaris aquaporins short-term regulatory mechanisms: 1) protection against dephosphorylation seems not to be necessary to keep water channels active after PMV isolation, 2) Pf is markedly reduce in the presence of calcium and 3) acidic pH is capable of shut-down water transport, being the pH sensor in the citoplasmatic side of the membrane. Supported by FONCYT PICT/99 5145 and IFS-Fundación Antorchas Grant C3052/2. References: 1. Maurel C, et al. 1997b Prod. Nat. Acad. Sci. USA. 94:7103-7108. 2. Niemietz, CM and Tyerman, S 1997 Plant Physiol. 115:561-567. 3. Gerbeau P et al, 1999 Plant J. 18:577-587. 4. Gerbeau P, et al. 2002 The Plant J. 30(1):71-81. 5. Dordas C, et al. 2000 Plant Physiol. 124:1349-1361